Erythropoiesis is the production of red blood cells which occurs to offset cell destruction. Erythropoiesis is a controlled physiological mechanism that enables sufficient red blood cells to be available for proper tissue oxygenation. Naturally occurring human erythropoietin (hEPO) is a glycoprotein containing 165 amino acids that is produced in the kidney and is the humoral plasma factor which stimulates red blood cell production. Human EPO stimulates the division and differentiation of committed erythroid progenitors in the bone marrow. Human EPO exerts its biological activity by binding to receptors on erythroid precursors. Naturally occurring human erythropoietin is an acidic glycoprotein present in low concentrations in plasma to stimulate replacement of red blood cells which are lost through ageing.
Erythropoietin has been manufactured biosynthetically using recombinant DNA technology (Egrie, J. C., et al., Immunobiol. 72 (1986) 213-224) and is the product of a cloned human EPO gene inserted into and expressed in the ovarian tissue cells of the Chinese hamster (CHO cells). Naturally occurring human EPO is first translated to a 166 aa containing polypeptide chain with arginine 166. In a postranslational modification, arginine 166 is cleaved by a carboxypeptidase. The primary structure of human EPO (165 aa and 166 aa) is shown in SEQ ID NO:1 and SEQ ID NO:2. There are two disulfide bridges between Cys7-Cys161 and Cys29-Cys33. The molecular weight of the polypeptide chain of human EPO without the sugar moieties is 18,236 Da. In the intact EPO molecule, approximately 40% of the molecular weight is accounted for by the carbohydrate groups (Sasaki, H., et al., J. Biol. Chem. 262 (1987) 12059-12076).
Because erythropoietin is essential in red blood cell formation, it is useful in the treatment of blood disorders characterized by low or defective red blood cell production. Clinically, EPO is used in the treatment of various ailments, for example, anemia in chronic renal failure patients (CRF) and in AIDS and cancer patients undergoing chemotherapy (Danna, R. P., et al., In: M B, Garnick, ed. Erythropoietin in Clinical Applications—An International Perspective. New York, N.Y.: Marcel Dekker; 1990, pp. 301-324). However, the bioavailability of currently available protein therapeutics such as erythropoietin is limited by their short plasma half-life and susceptibility to protease degradation. These shortcomings prevent them from attaining maximum clinical potency.
Modifications of the amino acid sequence of EPO have been disclosed, for example, in a number of references including U.S. Pat. No. 4,835,260; WO 94/25055; WO 94/24160; WO 94/02611; WO 95/05465.
Both human urinary derived erythropoietin and recombinant erythropoietin (expressed in mammalian cells) contain three N-linked and one O-linked oligosaccharide chains which together comprise about 40% of the total molecular weight of the glycoprotein. N-linked glycosylation occurs at asparagine residues located at positions 24, 38 and 83 while O-linked glycosylation occurs at a serine residue located at position 126 (Lai, et al., J. Biol. Chem. 261 (1986) 3116; Broudy, V. C., et al., Arch. Biochem. Biophys. 265 (1988) 329). The oligosaccharide chains have been shown to be modified with terminal sialic acid residues. Enzymatic treatment of glycosylated erythropoietin to remove all sialic acid residues results in a loss of in vivo activity but does not affect in vitro activity (Lowy et al., Nature 185 (1960) 102; Goldwasser, E., et al. J. Biol. Chem. 249 (1974) 4202-4206). This behavior has been explained by rapid clearance of asialoerythropoietin from circulation upon interaction with the hepatic asialoglycoprotein binding protein (Morrell et al., J. Biol. Chem. 243 (1968) 155; Briggs, D. W., et al., Am. J. Physiol. 227 (1974) 1385-1388; Ashwell, G., and Kawasaki, T., Methods Enzymol. 50 (1978) 287-288). Thus, erythropoietin possesses in vivo biological activity only when it is sialylated to avoid its binding by the hepatic binding protein.
The role of the other components in the oligosaccharide chains of erythropoietin is not well defined. It has been shown that partially diglycosylated erythropoietin has greatly reduced in vivo activity compared to the glycosylated form but does retain in vitro activity (Dordal, M. S., et al., Endocrinology 116 (1985) 2293-2299). In another study, however, the removal of N-linked or O-linked oligosaccharide chains singly or together by mutagenesis of asparagine or serine residues that are glycosylation sites sharply reduces in vitro activity of the altered erythropoietin that is produced in mammalian cells (Dube, S., et al., J. Biol. Chem. 263 (1988) 17516-17521).
Oligonucleotide-directed mutagenesis has been used to prepare structural mutants of EPO lacking specific sites for glycosylation (Yamaguchi, K., et al., J. Biol. Chem. 266 (1991) 20434-20439; and Higuchi, M., et al., J. Biol. Chem. 267 (1992) 7703-7709). Cloning and expression of non-glycosylated EPO in E.coli is described by Lee-Huang, S., Proc. Natl. Acad. Sci. USA 61 (1984) 2708-2712; and in U.S. Pat. No. 5,641,663.
EP 0 640 619 relates to analogs of human erythropoietin comprising an amino acid sequence which includes at least one additional site for glycosylation. The added sites for glycosylation may result in a greater number of carbohydrate chains, and higher sialic acid content, than human erythropoietin. Erythropoietin analogs comprising amino acid sequences which include the rearrangement of at least one site for glycosylation are also provided. Analogs comprising an addition of one or more amino acids to the carboxy terminal end of erythropoietin wherein the addition provides at least one glycosylation site are also included.
PEGylation of glycosylated EPO is described in WO 01/02017. Such molecules show an improved biological activity. WO 00/32772 and Francis, G. E., et al., Int. J. Hem. 68 (1988) 1-18, describe polyethylene glycol- modified non-glycosylated EPO. The molecules of WO 00/32772 are additionally modified at positions 166. Such molecules are described as not causing a significant increase in hematocrite. The PEG-polymer portion consists of 1-5 polymer chains. WO 00/32772 suggests to control the degree and site of PEGylation by lowering the pH and reducing the PEG: amine ratio. Reactions run at pH 7 and 1.5:1 molar ratio of PEG-aldehyde: amine groups, preferentially react with the N-terminal α-amino group.
In spite of the numerous modifications that are known for EPO, there still exists a need for further EPO muteins with modified properties, especially with modified clearance and simple, reproducible methods for its production.